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19.01.2010
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The production of 2H-labeled amino acids by a new mutant of RuMP fucultative methylotroph Вrevibacterium methylicum Oleg Mosin Julia
The production of 2 H-labeled amino acids by a new mutant of RuMP fucultative methylotroph В
revibacterium methylicum
Oleg V. Mosin 1
1
Department of Biotechnology, M. V. Lomonosov State Academy of Fine Chemical Technology, Vernadskogo Prospekt 86, 117571, Moscow, Russia
Summary
The biosynthesis of 2 H-labeled phenylalanine was done by converse of low molecular weight substrates ([U-
2 H]methanol and 2 H 2
O) in a new RuMP facultative methylotrophic mutant Brevibacterium methylicum. To make the process work, adapted cells with improved growth characteristics were used on minimal medium M9 with the maximum content of
2 H-labeled substrates. Alanine, valine, and leucine/isoleucine were produced and accumulated exogeneously in addition
to the main product of biosynthesis. Electron impact mass spectrometry of methyl esters of the N
-Dns-amino acid mixture obtained after the chemical derivatization of growth medium with dansyl chloride and diazomethane, was done to calculate the deuterium enrichment of the amino acids synthesized. The experimental data testified to the character of labeling of amino acid molecules as heterogeneous; however, high levels of deuterium enrichment were detected in all presented molecules - for phenylalanine the enrichment was six, leucine/isoleucine - 5.1, valine - 4.7, and alanine - 3.1 deuterium atoms.
Keywords: Brevibacterium methylicum - Heavy water - Biosynthesis - 2 H-Labeled amino acids - Phenylalanine - EIMS
Abbreviations: EI MS: electron impact mass spectrometry; TLC: thin layer chromatography; DNSCl: dansylchloride; DZM:



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